Due to lack of effective therapy, primary brain tumors are the focus of intense investigation of novel experimental approaches that use vectors and recombinant viruses. Therapeutic approaches have been both indirect, whereby vectors are used, or direct to allow for direct cell killing by the introduced virus. Promising therapies can be designed by targeting fundamental molecular defects of the glioma cells. The function of p16-Rb-E2F pathway is abnormal in most malignant gliomas and therefore constitutes a suitable target for anti-cancer therapies. We have previously generated a conditional replicating adenovirus, D24, unable to bind to and inactivate the retinoblastoma protein (Rb). This tumor-selective adenovirus is able to replicate in glioma cells but not in normal cells. Although, the adenovirus induces a potent cytopathic effect in vitro, its anti-cancer effect in vivo is less dramatic. In this project, we propose a series of modifications in the D24 adenovirus in order to render the oncolytic virus more efficient infecting and killing glioma cells in vivo. In addition, experiments will be designed to introduce the necessary modifications in the D24 adenovirus to increase its specificity and to control pharmacologically its replication and spread. In vivo cancer gene therapy approaches for gliomas based on adenoviral vector-mediated gene delivery and oncolytic adenoviruses can be limited by the suboptimal efficacy of adenoviruses to infect tumor cells. This issue is mainly due to deficiency of the primary adenoviral receptor on the tumor targets. To circumvent this deficiency, we propose the construction of a tumor-selective adenoviral targeted to a tumor cell marker. In this regard, RGD-related integrins are frequently overexpressed in gliomas. Furthermore, these integrins recognize the RGD peptide motif. On this basis, we will construct an adenoviral vector genetically modified to contain such a peptide within the HI loop of the fiber protein as a means to alter viral tropism. This RGD-D24 adenovirus should infect glioma cells in vivo with extraordinary efficiency, increasing dramatically the oncolytic power of the D24. In Specific Aim 1. We propose to characterize the anticancer effect of D24-RGD in vitro in comparison with D24. In Specific Aim 2, the D24-RGD construct will be characterized in vivo using an orthotopic glioma animal model. In addition, we will examine the correlation between the anti-cancer effect of the D24-RGD ant its spread throughout the tumor. The experiments will require pathological examination of the tumors, viral protein expression, as well as examination of spread of the virus throughout the tumor. We will asses the replication of the virus within the tumor and titer the viral production in vivo. Finally, we will analyze how the administration of anti-adenoviral drugs influence the growth of D24-RGD-infected tumors. In Specific Aim 3, we will combine a high-effective oncolytic adenovirus with a regulatory system that can be used to control viral replication in vivo in a selected site and at a desired time. The D24-RGD construct will be genetically modified to include drug response elements sensitive to the effect of tetracycline. To obtain tissue-specific expression of the target gene, we will coupled the regulator to a cancer specific (E2F-1) promoter to drive the early viral genes. The combination of an inducible system and a tissue-specific promoter will allow the development of an innovative oncolytic system, which is able to kill cancer cells and spread within the tumor in a cell type-specific and time- and level-controllable fashion.